A Novel and Rapid PCR-Based Method for Genotyping Human Papillomaviruses in Clinical Samples

Abstract

Many human papillomavirus (HPV) genotypes are associated with cervical carcinoma. We demonstrate the utility of an innovative technique for genotyping of HPV in cervical tissue samples. This method provides an accurate means of identification of the specific HPV genotypes present in clinical specimens. By using the MY09-MY11 and the GP5+-GP6+ consensus primer pairs, HPV sequences were amplified by nested PCR from DNA isolated from cervical smear samples. This led to the production of an approximately 140-bp PCR product from the L1 (major capsid) gene of any of the HPVs present in the sample. PCR was performed with a deoxynucleoside triphosphate mixture which resulted in the incorporation of deoxyuridine into the amplified DNA product at positions where deoxythymidine would normally be incorporated at a frequency of about once or twice per strand. Following the PCR, the product was treated with an enzyme mix that contains uracil N-glycosylase (UNG) and endonuclease IV. UNG removes the uracil base from the nucleotide, and endonuclease IV cleaves the phosphodiester bond at this newly formed abasic site, producing fragments of various sizes. By having end labeled one of the amplification primers, a DNA ladder which is analogous to a “T-sequencing ladder” was produced upon electrophoresis of the products. By comparing this T-sequencing ladder to the known sequences of HPVs, the genotypes of unknown HPV isolates in samples were assigned. Data showing the utility of this technique for the rapid analysis of clinical samples are presented.

Routine genotyping of human papillomavirus samples in Denmark

“…However, no difference between nested MY09/MY11 PCR plus GP5+/GP6+ and single MY09/MY11 PCR has also been observed (27). The idea of combining the primer set GP5/GP6 with the MY09/MY11 primer set in a nested PCR was conceived in 1995 by Evander and co‐workers (14), and this assay (more often with GP5+/GP6+ as the inner primer set) is now widely used for the detection of HPV (28, 29). Our results showed a 10‐fold increase in the sensitivity of the nested MY09/MY11 and GP5+/GP6+ PCR as compared to single MY09/MY11 PCR.…”

Sentinel-base DNA genotyping using multiple sequencing primers for high-risk human papillomaviruses

“…For genotyping purposes, these assays have been combined with a variety of hybridization techniques, including dot blots [11,12], reverse hybridization line probe assays [13,14], Tladder generation [15], and DNA microarray [16,17]. A general drawback of hybridization techniques is that they may result in cross-hybridizations of closely related genotypes as well as non-specific hybridization.…”

A role for arrays in clinical virology: fact or fiction?

“…Target Reference Papillomaviruses L1 region (Coutlee et al, 2002;Kleter et al, 1998;Nelson et al, 2000) Enteroviruses 5 -NCR (Clewley, 1995;Shih et al, 2003;Zoll et al, 1992) Herpesviruses DNA polymerase (Calvario et al, 2002;Ehlers et al, 1999;Minjolle et al, 1999;VanDevanter et al, 1996;Yamamoto and Nakamura, 2000) Retroviruses…”